Lactobacillus LB is a postbiotic generated following fermentation by Limosilactobacillus fermentum and Lactobacillus delbrueckii. Lactobacillus LB alleviates acute diarrhoea and ameliorates the symptoms of irritable bowel syndrome. Here, we investigated whether modulation of intestinal ion transport and motility contributes to these beneficial effects and whether the postbiotic produced with both strains contributes to a unique biophysiological profile. In Ussing chamber studies, low lactose-Lactobacillus LB (LL-LB) significantly increased baseline short-circuit current, and this was partially mediated by sodium-D-glucose transporter 1. In organ baths, LL-LB significantly decreased ileal tone and increased carbachol-induced contractility. Relative to LL-LB, preparations produced using a single strain fermentate generated from L. fermentum significantly increased baseline short-circuit current and inhibited carbachol-induced contractility. Our data demonstrate a unique biophysiological profile for the dual strain postbiotic and support a direct and immediate effect of LL-LB on host physiology ex vivo which could contribute to the clinical efficacy of Lactobacillus LB.